Natural Products Chemistry & Research

Harmful marine algae are widely spread throughout the coastal areas around many continents and have insecticidal value [1]. Liagora ceranoides J.V. Lamouroux (Liagoraceae: Rhodophyta) is a macroscopic marine red algal seaweed which is heavily calcified, pinkish in color and ramiform with cylindrical and dichotomous branches [2]. Spodoptera litura (Fab.) is a polyphagous pest of cosmopolitan distribution which comes out as a serious and dominant pest in agricultural and horticultural crops [3]. Indiscriminate use of synthetic pesticides caused serious ill effects to the precious mother earth, offers pest resistance and harming beneficial organisms viz., predators, parasitoids and pollinators [4]. Hence, switching over to eco-friendly insect pest management considered to be a better alternative. Among different alternatives, seaweeds offer a non-toxic way to manage the pests. Hence, the present study dealt with testing the bio-efficacy of methanol extracts of L. ceranoides against growth and development of S. litura .

Collection of seaweeds

Seaweed belonging to Rhodophyta, Red algae, Liagora ceranoides was collected from Rameswaram coast, Tamil Nadu, India. The seaweed collected by hand picking method was immediately washed with fresh sea water and then carefully washed thoroughly three times with fresh water to remove the excess salt, sand and epiphytes to drain off the water, the algae were wiped with a blotting sheet and air dried under shade for a fortnight and stored [1]. The sea weeds were identified at Parangipettai, Faculty of Marine Biology, Annamalai University, Tamilnadu, India.

Mass culturing of Spodoptera litura

Spodoptera litura was mass cultured on natural diet castor leaves under laboratory conditions in sterilized plastic buckets and the required amount of third instar larvae was used for the bio-assay experiments.

Preparation of seaweed solvent extract

Fifteen gram of partially powdered red algal seaweed L. ceranoides was packed and loaded in Soxhlet apparatus (GI-1706 A) individually and refluxed with Methanol for 12 hours continuously. The extracted solvents were evaporated in a hot plate. The final extract was elucidated with methanol and used for the evaluation experiments. The extracts were stored at -20°C [5].

Prepared L. ceranoides solvent extracts were treated to the uniform aged third instar larva @1, 10, 30, 50, 100, 200 μl/L with solvent and water control. The methanol extracts were tested against a homogenous culture (third instar larva) of Spodoptera litura using leaf dip bio-assay. To compare the performance of solvent effect on the larva, a control was maintained along with an absolute control, replicated thrice under completely randomized block design. Data on repellency, antifeedant, larval mortality, pupation, pupal malformation, adult malformation and adult emergence were recorded, and the data were statistically analysed and the results were presented [6].

GC-MS analysis

Preparation of extract: The seaweed was shade dried and powdered with the help of electric blender. The sample was extracted with ethanol and 2 μl of this solution was employed for GC-MS analysis.

GC condition and identification of compounds: The seaweed powder was extracted with ethanol and 2 μl of this solution was employed for GC-MS analysis. The GC-MS analysis was carried out with 30 m × 0.25 mm ID × 0.25 μm df with column BR-5 MS (5% Diphenyl/95% Dimethyl poly siloxane). The instrument was set to an initial temperature of 110°C and maintained at this temperature for 3.50 minutes. At the end of this period, the oven temperature was rose up to 280°C, at the rate of an increase of 50 C/min, and maintained for 12 min. Injection port temperature was ensured as 280°C. The ionisation voltage was 70 eV. Mass spectral scan range was set at 50-500 (m/z) amu. Using computer searches on a NIST Ver-11 MS data library and comparing the spectrum obtained through GC-MS, compounds present in the seaweed sample were identified with their compound name, chemical formula and molecular weight [7].

The results revealed that the larval mortality initiated from day one and increased up to 5th day. Among the sea weed treatments, maximum mortality (80.00%) was exhibited at 200 μl/L concentration followed by 100 and 50 μl/L concentration (73.33 and 66.66%). No larval mortality was observed in solvent control and control in the same period of observation (Table 1).

Table 1: Influence of red algal seaweed Liagora ceranoides methanol extract on the developmental stages of Spodoptera litura.

Observed pupation was minimum in 200 μl/L concentration (20.00%) and maximum in 1 μl/L (53.33%) in L. ceranoides with cent percent pupation in control. Pupal malformation was 13.33 per cent at 200 μl/L concentration. The pupal mortality and pupal malformation was 6.66 and 13.33 per cent at 200 μl/L concentration which was higher than the other treatments and control. Cinnamomum camphora oil extracts are evaluated against S. litura and reduction in feeding, pupation and adult emergence at 2.5 and 2.0 mg/larva concentration have been observed [8]. There was no adult emergence at 200 μl/L concentration i.e., the pupal to adult conversion ratio was 1:0.00 whereas complete emergence was noticed at 1 μl/L concentration (1:1.00) in both the seaweeds. The results obtained in this study are in accordance with the reports wherein they have observed that the methanol extract of Ulva fasciata and Chaetomorpha antennina (green algal seaweed) have showed increased larval mortality and growth inhibitor activity against S. litura . Methanol extract of Thevetia nerifolia leaves at 2.5 per cent concentration demonstrate 53.8 and 29.6 per cent larval mortality and pupation of S. litura respectively [6]. The pupation of the insect have been reduced significantly in all the concentrations and fractions of hexane, chloroform, ethyl acetate and methanol fractions [9].

In this study, it has been observed that the sea weed used for evaluating their potential against test insect was well supported by many evidences from other plants, green algae and red algae against many insects as follows.

Methanolic extract of Gracilaria crassa and Hypnea Valentia demonstrate higher level larval mortality of A. aegypti [10]. The chloroform extract of Calotropis procera exhibits the best larvicidal activity against S. litura [11]. Crude chloroform extract and emulsifiable concentrate (EC) of seaweed Caulerpa scalpelliformis repel S. litura and Dysdercus cingulatus in dose dependent manner [5]. Methanol extracts from different marine algae Caulerpa racemosa , C. scalpelliformis , U. fasciata , Padina tetrastromatica , Stoechospermum polypodioides , Sargassum wightii , Cheilosporum spectabile and Gracillaria edulis are effective on the root knot nematode (Melodogyne javanica ) [12].

The GC-MS-MS studies of L. ceranoides has shown many phytochemicals which contribute to various biological activity. The active principles, with their retention time (RT), molecular formula, molecular weight (MW), concentration (peak area%) and the molecular structure are presented in Table 2. The GC-MS Chromatogram of the number of peaks of the compounds detected was shown in Figure 1. The total number of compounds identified in ethanol extract of L. ceranoides was 13[13-16].

Figure 1: GC-MS chromatogram of Liagora ceranoides .

Table 2: Phytochemical in red algal seaweed Liagora ceranoides (Based on GC-MS analysis).

The volatile oils of L. ceranoides displayy insecticidal activity on stored product pests viz., Oryzeaphilus mercator , Tribolium castaneum due to the presence of principle bioactive compounds viz., Ethyl cinnamate, Tetradecane, Pseudoionone and methyl tetradeconate. Oleic acid is responsible for the mosquito repellent activity. Nonadecane act as a pheromone and this attracts the parasitoid of the host insect. Benzyldiethyl- (2,6-xylylcarbamoylmethyl)- ammonium benzoate called denatonium benzoate present in G. corticata has been used as toxic bait for pestiferous German wasp (Vespula germanica ). The toxic effect of L. ceranoides on S. litura may be due to the presence of any one or more than one phytochemical identified and its characterization in future would pave for many insights in developing a potential biopesticide for future.

The authors are grateful to Tamilnadu State Council for Science and Technology (TNSCST), Govt. of Tamilnadu, India, for extending financial support for conducting the research. The authors are also grateful to Department of Entomology, Faculty of Agriculture, Annamalai University, India for providing laboratory facilities.